PROJECT SUMMARY/ABSTRACT Although we have a great understanding on the physiological importance of the biofilm matrix as a whole, we know relatively little about the mechanistic role of the individual components and how they interact to form structures at the micron-scale. Here, we utilize a simple model system that naturally produces striking structures to dissect the individual and collective roles of extracellular secretions in structure formation. Aging colonies of Pseudomonas fluorescens produce spatially isolated patches exclusively through mutations that reduce or remove the function of a post-transcriptional repressor (RsmE). RsmE homologs are widely spread among bacteria and prevent the translation of cognate mRNA to regulate diverse traits, including the production of extracellular secretions and biofilm formation. At the micron-scale, clonal aggregates of rsmE mutants expand space by pushing away and preventing the encroachment of the parent cells, dramatically reducing the local density within the clonal patch. Importantly, rsmE mutants neither show growth advantage in pure culture nor produce toxic compounds, and the evolutionary advantage requires the physical presence of the parent cells. We thus hypothesize that RsmE-regulated secretions function collectively to form spatial structures that solve the problem of crowding in a densely populated colony. To address this, we will: (1) identify the components of the RsmE-secretome through transposon mutagenesis, mass spectrometry, and RIP-seq to disrupt and identify secretion genes that prevent/reduce patch formation; mass spectrometry to directly identify the secretions; RIP- seq to identify mRNA that bind directly to RsmE; (2) characterize the functional role of RsmE-secretions in structure formation through competitions and confocal microscopy; (3) identify the key amino acid residues of RsmE that define its functional specificity by competing naturally derived missense mutants against one another, RIP-seq and microscopy of to assess the loss of mRNA-specificity and spatial consequences, respectively, that are associated with substitutions in the key residues, and RIP-seq to identify mRNA that bind exclusively to RsmE and those that bind promiscuously to two distinct paralogs of RsmE that are highly conserved in amino acid sequence. At the completion of this project, we will demonstrate (1) how a change to a single nucleotide could drive complex multicellular behaviors that are not predictable based on the attributes of the individuals in isolation, (2) how extracellular secretions function individually and collectively at the micron-scale to capture space and optimal positioning, and (3) how RsmE manifests specificity to change the general perception that Rsm-paralogs are functionally redundant. Particularly, the convergence of these individual outcomes onto spatial structures will represent a unique and substantial contribution to advance the field. We will also demonstrate the power and utility of harnessing and integrating natural selection in the lab as an analytical tool to characterize the function of a pleiotropic regulator. This proposal emphasizes AREA?s main objective of training and mentoring undergraduate students to directly carry out meritorious research toward careers in science and medicine.